Bi-functional alkylating agents are important components of many chemotherapeutic regimens that form a variety of DNA damages including mono-adducts, intra- and interstrand DNA cross- links (ICL). Interstrand cross-links are among the most deleterious lesions reminiscent of DNA double strand breaks, because they constitute absolute blockades to DNA replication, recombination, and gene transcription. While mechanisms for repairing mono-adducts and intrastrand cross-links have been studied extensively, little is known about the components and the mechanisms of interstrand cross-linking repair in humans due to the lack of lesion-specific assays. We have developed reporter reactivation-based approaches to examine specifically the repair processes of DNA cross-links in mammalian cells. Our hypothesis is that both homology-dependent and -independent mechanisms may be involved in the processing of DNA interstrand cross-links. Our preliminary studies have provided evidence for a recombination-independent mechanism for ICL removal in human cells. Mutation analysis also suggested the error-proneness of this pathway. In the proposed study we will further examine the validity of this hypothesis by identifying gene products involved in the recombination-independent repair of ICLs and establishing a possible mechanism for this pathway. Mutations spectrum of this error-prone repair mechanism will be characterized. Effects of DNA replication and transcription on the mutagenic ICL repair pathway will be examined. Homologous recombination, stimulated by ICL repair, will be tested in an established assay system to provide molecular and genetics evidence for ICL-stimulated sister chromosome exchange. These studies should generate informative results on the repair mechanisms of DNA interstrand cross-links in mammalian cells and allows us to better understand the mutagenic consequences of DNA alkylating chemotheraputic agents.